Protein kinases involved in the endothelial nitric oxide synthase activation by beta-adrenoceptors stimulation in human umbilical vein endothelial cells
- VernacularTitle:β受体激动增加人脐静脉内皮细胞内皮型一氧化氮合酶活性的细胞内机制
- Author:
Juanjuan PU
;
Biao XU
- Publication Type:Journal Article
- From:
Chinese Journal of Tissue Engineering Research
2008;12(33):6589-6592
- CountryChina
- Language:Chinese
-
Abstract:
BACKGROUND: Beta-adrenergic activation can enhance signal-transduction pathway of endothelial nitric oxide (NO). Vascular endothelial NO production in response to β -adrenergic activation is important in the normal control of vessel tone by the sympatheadrenal system. Previous trials have demonstrated that endothelial nitric oxide synthase (eNOS) activated by isoprenaline, β -adrenergic agonist, may be correlated with phosphorylation of eNOS. OBJECTIVE: To investigate the possible protein kinase involved in the signal transduction of the eNOS activation by β -adrenoceptor (β AR) stimulation in human vascular endothelium. DESIGN: Comparative observation. SETTING: Department of Geriatrics, First Affiliated Hospital of Zhengzhou University. MATERIALS: The experiment was performed at the Open Key Clinic Medical Experimental Laboratory of University of Henan Province between September 2006 and June 2007. Fresh umbilical cords were obtained following delivery of healthy babies to healthy normotensive mothers, either by vaginal delivery or by elective Caesarean section. The written informed consent was obtained from all donors. Approval was granted by the Affiliated Hospital of Zhengzhou University. Isoprenaline, H-89, Wortmannin, [3H]-L-arginine were from Sigma, USA. METHODS: Blank control group, H-89 group (protein kinase inhibitor), Wortmannin group (phosphatidylinositol 3-kinase inhibitor) and H-89+Wortmannin group were set up, and isoprenaline subgroup and blank subgroup were subdivided with 3 tubes in each group. Human umbilical vein endothelial cells (HUVECs) were cultured with H-89 and Wortmannin, separately for 10 minutes, then agonist isoprenaline or vehicle was added and the incubation continued for another 30 minutes, eNOS activity was determined by the conversion of [3H]-L-arginine to [3H]-L-citrulline. The results were corrected by protein concentration in the corresponding cellular extracts. MAIN OUTCOME MEASURES: Changes in eNOS activity of each group. RESULTS: ①Basal [3H]-L-citrulline production was (3 085.62±272.72) mBq per 1 μ g protein, lsoprenaline increased [3H]-L-citrulline formation by (30.72 ± 3.91 )% compared with the control group (P < 0.001 ). H-89 or Wortmannin alone did not alter eNOS activity [(0.44± 1.00)%, (0.36±1.47)%, P > 0.051. ②The increase of eNOS activity by isoprenaline wassignificantly inhibited by pretreatment of H-89 and Wortmannin [(4.73±2.19)%, (17.35±3.72)%, P < 0.001]. ③The inhibition of protein kinase and phosphatidylinositol 3-kinase to isoprenaline-induced increase in eNOS activity [(4.92± 0.99)%] was similar to protein kinase alone, but not in an additive effect (P > 0.05). CONCLUSION: Protein kinase and phosphatidylinositol 3-kinase are involved in the signal transduction of β- adrenoceptors mediated eNOS activation.
